Previous studies have shown that GST-MDA-7 reduces proliferation and causes tumor cell– and transformed cell–specific killing and radiosensitization in malignant glioma and prostate cancer cells. However, although JNK signaling plays a key role in radiation-enhanced killing by mda-7/IL-24 of tumor cells, the precise signaling pathways provoked by GST-MDA-7 as a single agent and casually related to its cancer-specific cell killing effects in human renal carcinoma cells are still not well understood. The studies in this article were designed to clarify these issues and define how RCCs respond to GST-MDA-7 exposure and how, mechanistically, alterations in multiple signaling pathways affect their cell viability.
A GST-MDA-7 concentration that caused profound toxicity ~72 hours after exposure in RCCs correlated with strong activation of the JNK1/2 and p38 MAPK pathways. This treatment, in parallel, nearly abolished ERK1/2 signaling. Multiple studies using a variety of cytokine and toxic stimuli document that prolonged JNK1/2/3 and/or p38 MAPK activation in a wide variety of cell types can trigger cell death (34
). The balance between the readouts of ERK1/2 and JNK1/2/3 signaling may also represent a general key homeostatic mechanism that regulates cell survival versus cell death processes (34
). Inhibition of either JNK1/2 or p38 MAPK reduced GST-MDA-7 toxicity, and inhibition of both pathways abolished cell killing. Prior work, treating primary hepatocytes with low doses of bile acids, which cause ligand-independent activation of CD95, discovered that JNK1/2 activation was CD95 dependent, and our present studies showed that JNK1/2 and, to a lesser extent, p38 MAPK activation following GST-MDA-7 treatment also required CD95 signaling. CD95 signaling in GST-MDA-7–treated renal carcinoma cells was responsible, in large part, for PERK activation, and expression of dominant negative PERK blocked MDA-7/IL-24–induced JNK1/2 and p38 MAPK activation as well as the inactivation of ERK1/2. Matsuzawa et al. (36
) have previously implicated a tumor necrosis factor receptor–associated factor 2/apoptosis signal–regulating kinase 1/JNK cascade downstream of inositol-requiring enzyme 1 in endoplasmic reticulum stress responses in multiple cell types, and based on our data, PERK-dependent signaling could also feed into this cell survival regulatory process. Collectively, in RCCs MDA-7/IL-24, lethality is reliant on CD95-PERK–dependent JNK1/2 and/or p38 MAPK signaling.
In one ovarian cancer cell line, MDA-7/IL-24, lethality was mediated by CD95-caspase-8 signaling, indicating that the extrinsic pathway to apoptosis could be activated by this cytokine (18
). Overexpression of c-FLIP-s or the caspase-8 inhibitory protein CRM A blocked GST-MDA-7 lethality. Knockdown of CD95 or FADD expression similarly reduced GST-MDA-7 toxicity in RCCs. Hence, as was observed in one ovarian line, our findings in RCCs argue that MDA-7/IL-24 toxicity requires death receptor-caspase-8 signaling. However, in all of our prior work with MDA-7/IL-24 in breast, prostate, pancreatic, and brain tumor cells, as well as from the work of others, it was noted that MDA-7/IL-24–induced cell death was mediated by disruption of mitochondrial function with little or no involvement of death receptors or caspase-8 in the killing process. For example, in LNCaP prostate cancer cells, the toxicity of Ad.mda-7
, either as an individual agent or when combined with a reactive oxygen species–inducing treatment such as ionizing radiation exposure, has been linked to changes in mitochondrial function (17
). MDA-7 expression resulted in altered ratios in the expression of proapoptotic BH3 domain–containing proteins, such as BAX, and antiapoptotic proteins, such as BCL-2 and BCL-XL, with the subsequent release of cytochrome c
into the cytosol followed by activation of caspase-9 and caspase-3 (9
In other cell types that lack expression of BAX, such as DU145, Ad.mda-7
is an even more potent inducer of tumor cell death than is observed in LNCaP cells. In glioblastoma cells, our data argued that at least five BH3 domain–containing proteins could potentially mediate GST-MDA-7 toxicity downstream of GST-MDA-7–stimulated activation of PERK and JNK1–JNK3 and subsequent reduction in BCL-XL levels (22
). Our data in RCCs showed that in addition to GST-MDA-7 reducing c-FLIP-s expression, PERK signaling also leads to reduced expression of the short-lived mitochondrial protective protein MCL-1. Collectively, based on these findings, it is tempting to speculate that the reason why multiple transformed cell types exhibit MDA-7/IL-24 toxicity regardless of genetic background is the pleiotropic range of proapoptotic proteins and pathways that can be recruited by this cytokine to initiate mitochondrial cell death processes.
We have recently published several studies arguing that ligand-independent CD95 signaling via PERK promoted both a caspase-8 death signal as well as a counteracting PERK-dependent protective autophagy signal (29
). Others have found that inhibition of caspase-8 can promote a toxic form of autophagy and that the death domain of FADD can promote a toxic form of autophagy when associated with ATG5 in nontransformed cells (37
). There are three primary unfolded protein response sensors: PERK, activating transcription factor 6, and inositol-requiring enzyme 1. As unfolded proteins accumulate, BiP (Grp78), the heat shock protein-70 endoplasmic reticulum–resident chaperone, dissociates from PERK, activating transcription factor 6, or inositol-requiring enzyme 1. BiP/Grp78 dissociation from PERK allows this protein to dimerize, autophosphorylate, and then phosphorylate eukaryotic translation initiation factor α, the protein required for bringing the initiator methionyl-tRNA to the 40S ribosome (refs. 29
, and references therein). Phosphorylated eIF2α thus leads to repression of global translation, helping to allow cells to recover from the accumulation of unfolded proteins. Reduced translation, however, can also lower expression of some pro-survival proteins such as MCL-1 and c-FLIP-s, leading to increased cell death (29
). In glioma, MDA-7/IL-24 induced PERK-dependent autophagy in a manner not dependent on death receptor or caspase-8 signaling (22
). GST-MDA-7–induced autophagy in RCCs was significantly, but not fully, suppressed by knockdown of CD95. MDA-7/IL-24 binds to Grp78/BiP, and in glioblastoma cells we initially assumed this binding would play a central role in regulating PERK activity/activation and in causing elevated levels of toxic autophagy, particularly because we subsequently noted that BiP/Grp78 overexpression suppressed MDA-7/IL-24–induced autophagy and cell killing (23
). More recently, we also found that overexpression of BiP/Grp78 in RCCs suppressed GST-MDA-7–induced autophagy.11
Thus, in glioblastoma cells the autophagy being observed after MDA-7/IL-24 treatment is dependent on BiP/Grp78 dysregulation (majority), whereas in RCCs it seems that the autophagy being observed after MDA-7/IL-24 treatment is dependent on both CD95 signaling (majority) and dysregulation of BiP/Grp78 (minority).
In glioblastoma cells, we noted that knockdown of either Beclin1 or ATG5 expression abolished GST-MDA-7–induced autophagy and reduced cell killing. In RCCs, knockdown of either Beclin1 or ATG5 expression also abolished GST-MDA-7–induced autophagy, and knockdown of ATG5 suppressed, but did not abolish, apoptosis. Whether the autophagy induced by GST-MDA-7 through CD95 or through BiP/Grp78 binding represents different survival or toxic signals will need to be defined in a subsequent report. A significant difference between GST-MDA-7–induced cell killing in RCCs and glioblastoma cells was the involvement of cathepsin proteases. In glioblastoma cells, cathepsin proteases were essential for MDA-7/IL-24 lethality, whereas in RCCs, inhibition of cathepsin activity caused only a weak often nonsignificant trend toward lower MDA-7/IL-24 toxicity. It has been shown by many groups that cathepsin proteases play a central role in the biology of glioblastoma, and elevated expression of cathepsin proteases together with reduced expression of cystatins correlates with increased glioblastoma invasiveness and patient morbidity. In contrast, in RCCs expression of cathepsin D has been correlated with an improved long-term patient survival, and cathepsin serum levels do not predict patient disease staging or tumor load (39
). These findings again suggest that a reason why MDA-7/IL-24 kills so many transformed cell types, regardless of genetic background, is the diverse range of proapoptotic signals that are recruited by this cytokine to initiate cell death processes.
In our studies, in treating primary hepatocytes with bile acids or RCC/hepatocellular carcinoma/pancreatic tumor cells with the drugs sorafenib and vorinostat, we discovered that ligand-independent activation of CD95 was dependent, in part, on the actions of ASMase and the de novo
ceramide synthesis pathway (29
). In RCCs, but not glioblastoma cells, GST-MDA-7 caused a ligand-independent activation of CD95 as judged by death-inducing signaling complex formation and CD95 surface localization. In a similar manner to our data with sorafenib and vorinostat in RCCs using siASMase and myriocin, knockdown of ASMase or ceramide synthase-6 expression or treatment with myriocin reduced CD95 activation and reduced cell killing. Recent studies have suggested that the de novo
and ASMase pathways of ceramide generation can cooperate to regulate lipid raft function (40
). Because ceramide synthase 6 and BiP/Grp78 are both endoplasmic reticulum–localized proteins, it is possible that GST-MDA-7, in some manner, by altering endoplasmic reticulum homeostasis, increases ceramide synthase-6 activity, which in RCCs leads to activation of CD95. Understanding how MDA-7/IL-24 regulates ceramide synthase gene function, ceramide levels, and the roles this lipid may play in the biology of MDA-7/IL-24 will require additional investigations.
At present, MDA-7 is under clinical investigation in phase II trials delivered as a form of gene therapy, Ad.mda-7
(ING241). In phase I studies in melanoma patients, Ad. mda-7
generated a significant number of partial and complete responses in patients with metastatic disease, arguing that this cytokine may also promote a vaccination effect in some tumors (12
). Because RCC is also known to be responsive to immunomodulatory cytokines, there is a possibility that expression of MDA-7 in vivo
could promote a vaccination effect in kidney cancer in addition to a tumor-specific cell killing effect. One issue for the use of Ad.mda-7
(ING241) in renal cancer is that RCCs are largely refractory to infection by type 5 adenovirus, and the development of a tropism-modified adenovirus to express MDA-7 in kidney cancer cells will be required. In conclusion, the present studies offer promise for expanded applications of MDA-7/IL-24 as a generalized and effective therapeutic for RCCs and potentially other cancers. Efforts are being expended to achieve these objectives and also to define clinically useful agents, such as arsenic trioxide or geldanamycins such as 17-N
-allylamino-17-demethoxygeldanamycin, which, when combined with MDA-7/IL-24, will increase therapeutic benefit for cancer patients.